Method and system of identifying and quantifying antibody fragmentation

ABSTRACT

Methods and system for identifying and quantifying antibody fragments and identifying the site of fragmentation on an antibody are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional Application No. 62/793,004, filed Jan. 16, 2019, which isherein specifically incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention generally pertains to a method and system for identifyingand quantifying antibody fragments and identifying the site offragmentation on an antibody.

BACKGROUND

Protein based biopharmaceutical products have emerged as important drugsfor the treatment of cancer, autoimmune disease, infection andcardiometabolic disorders, and they represent one of the fastest growingproduct segments of the pharmaceutical industry.

Protein digestion, either enzymatically or non-enzymatically, is animportant tool in protein identification, characterization, andquantification. The site of fragmentation can depend on the proteincomplexity and/or the method of digestion. In order to detect the siteof the fragmentation, identification of the clipped fragments isrequired.

Further, protein based biopharmaceuticals must meet very high standardsof purity. Proteins are susceptible to cleavage of the peptide backboneinto fragments, which can be catalyzed by acidic conditions used duringprocessing, handling, or storage. These clipped fragments could exhibita different mode of action and potential toxicity or immunogenicitycompared to the product. In addition, they can have a lower stabilitythan the product, which presents a higher risk for aggregation andimmunogenicity. Despite recent advances, it remains a challenge todevelop purity assay methods for quantitative evaluation of such clippedfragments. Therefore, it is important to monitor and characterize suchclipped fragments during different stages of drug development andproduction.

Analytical method for assays for detection of fragments should displaysufficient accuracy and resolution to detect and quantify the desiredproduct. Evaluation can be difficult due to similarities betweenstructural and physicochemical properties of the protein and the clippedfragment(s). Direct analysis can require isolation of the clippedfragment(s) in a sufficiently large amount for the assay, which can beundesirable and has only been possible in selected cases.

There is a long felt need in the art for a method and/or system foridentifying and quantifying antibody fragments and identifying the siteof fragmentation on an antibody.

SUMMARY

Growth in the development, manufacture and sale of protein-basedbiopharmaceutical products has led to an increasing demand forcharacterizing fragments of a protein and site of fragmentation of aprotein.

Exemplary embodiments disclosed herein satisfy the aforementioneddemands by providing methods and systems for identifying and quantifyingantibody fragments and identifying the site of fragmentation on anantibody.

This disclosure, at least in part, provides a method for quantifying afragment of an antibody in a sample.

In one exemplary embodiment, the method for quantifying a fragment of anantibody can comprise contacting the sample to a chromatographic systemhaving a mixed-mode size-exclusion chromatography resin with anadditional functionality, washing the mixed-mode size-exclusionchromatography resin using a mobile phase to provide an eluent includingthe fragment, and quantifying an amount of the fragment in the eluentusing a mass spectrometer.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with a hydrophobic interaction functionality

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with charge-charge interaction functionality.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting about 10 μg to about100 μg of a sample to a chromatographic system having a mixed-modesize-exclusion chromatography resin with an additional functionality.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase to provide aneluent including the fragment. In a specific aspect of this embodiment,the method for quantifying a fragment of an antibody in a sample cancomprise washing the mixed-mode size-exclusion chromatography resinusing a mobile phase that can be compatible with a mass spectrometer. Inanother specific aspect, the method for quantifying a fragment of anantibody in a sample can comprise washing the mixed-mode size-exclusionchromatography resin using a mobile phase, wherein the mobile phase canbe selected from ammonium acetate, ammonium bicarbonate, or ammoniumformate, or combinations thereof.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase containing upto 600 mM total salt concentration.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase with a flowrate of 0.2 ml/min to 0.4 ml/min.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the fragment can be adegradation product of the antibody.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the fragment is animpurity.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is amonoclonal antibody.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is atherapeutic antibody.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is abispecific antibody.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is amultispecific antibody.

In one aspect of this embodiment, the method for quantifying a fragmentof an antibody in a sample can comprise quantifying an amount of thefragment in said eluent using a mass spectrometer, wherein the massspectrometer can be a tandem mass spectrometer.

This disclosure, at least in part, provides a method for identifying afragment of an antibody in a sample.

In one exemplary embodiment, the method for identifying a fragment of anantibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, washing the mixed-modesize-exclusion chromatography resin using a mobile phase to provide aneluent including the fragment, determining the molecular weight of thefragment in the eluent using a mass spectrometer, and correlating themolecular weight data of the fragment to data obtained from at least oneknown protein standard.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting said sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with a hydrophobic interaction functionality

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting said sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with charge-charge interaction functionality.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting about 10 μg to about100 μg of a sample to a chromatographic system having a mixed-modesize-exclusion chromatography resin with an additional functionality.

In one aspect of this embodiment, method for identifying a fragment ofan antibody in a sample can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase to provide aneluent including the fragment.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase that can becompatible with a mass spectrometer. In a specific aspect, the methodfor identifying a fragment of an antibody in a sample can comprisewashing the mixed-mode size-exclusion chromatography resin using amobile phase, wherein the mobile phase can be selected from ammoniumacetate, ammonium bicarbonate, or ammonium formate, or combinationsthereof. In another specific aspect, the method for method foridentifying a fragment of an antibody in a sample can comprise washingthe mixed-mode size-exclusion chromatography resin using a mobile phasecontaining up to 600 mM total salt concentration.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase with a flowrate of 0.2 ml/min to 0.4 ml/min.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the fragment is adegradation product of the antibody.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is amonoclonal antibody.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is atherapeutic antibody.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is abispecific antibody.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the antibody is amultispecific antibody.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise contacting the sample to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality, wherein the fragment can be adigestion product of the antibody.

In one aspect of this embodiment, the method for identifying a fragmentof an antibody in a sample can comprise quantifying an amount of thefragment in said eluent using a mass spectrometer, wherein the massspectrometer can be a tandem mass spectrometer.

The disclosure, at least in part, provides a method for identificationof a site of fragmentation of an antibody.

In one exemplary embodiment, the method for identification of a site offragmentation of an antibody can comprise contacting a sample includingfragments of an antibody to a chromatographic system having a mixed-modesize-exclusion chromatography resin with an additional functionality,washing the mixed-mode size-exclusion chromatography resin using amobile phase to provide an eluent, determining molecular weight data ofthe fragments of the antibody in said eluent using a mass spectrometer,and correlating the molecular weight data of the fragments to dataobtained from at least one known protein standard.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with a hydrophobicinteraction functionality

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with charge-chargeinteraction functionality.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting about 10 μgto about 100 μg of a sample including fragments of an antibody to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with charge-chargeinteraction functionality and washing the mixed-mode size-exclusionchromatography resin using a mobile phase to provide an eluent includingthe fragments.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase that can becompatible with a mass spectrometer. In a specific aspect, the methodfor identification of a site of fragmentation of an antibody cancomprise washing the mixed-mode size-exclusion chromatography resinusing a mobile phase, wherein the mobile phase can be selected fromammonium acetate, ammonium bicarbonate, or ammonium formate, orcombinations thereof. In another specific aspect, the method foridentification of a site of fragmentation of an antibody can comprisewashing the mixed-mode size-exclusion chromatography resin using amobile phase containing up to 600 mM total salt concentration.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise washing the mixed-modesize-exclusion chromatography resin using a mobile phase with a flowrate of 0.2 ml/min to 0.4 ml/min.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the antibody can be a monoclonal antibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the antibody can be a therapeutic antibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the antibody can be a bispecific antibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the antibody can be a multispecific antibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the sample contains more than two fragments.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the fragments are formed due to degradation ofthe antibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the fragments are digestion products of theantibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise contacting a sampleincluding fragments of an antibody to a chromatographic system having amixed-mode size-exclusion chromatography resin with an additionalfunctionality, wherein the fragments are formed due to degradation ofthe antibody.

In one aspect of this embodiment, the method for identification of asite of fragmentation of an antibody can comprise identifying thefragment in said eluent using a mass spectrometer, wherein the massspectrometer can be a tandem mass spectrometer.

This disclosure, at least in part, provides a mixed mode chromatographicsystem.

In one exemplary embodiment, the chromatographic system can comprise achromatographic column having a having a mixed-mode size-exclusionchromatography resin with an additional functionality and a massspectrometer.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mixed-mode size-exclusion chromatography resin withhydrophobic interaction functionality.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mixed-mode size-exclusion chromatography resin withcharge-charge interaction functionality.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mixed-mode size-exclusion chromatography resin with anadditional functionality, which can be used for elution of about 10 μgto about 100 μg of a sample.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mixed-mode size-exclusion chromatography resin capable ofreceiving a mobile phase.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mixed-mode size-exclusion chromatography resin furthercapable of receiving a sample having a fragment.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mixed-mode size-exclusion chromatography resin capable ofbeing washed with a mobile phase.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a mass spectrometer coupled to a chromatographic columnhaving a mixed-mode size-exclusion chromatography resin with anadditional functionality.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a tandem mass spectrometer.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a chromatographic column having a having a mixed-modesize-exclusion chromatography resin with an additional functionality,wherein the mixed-mode size-exclusion chromatography resin can becompatible with a mobile phase selected from ammonium acetate, ammoniumbicarbonate, or ammonium formate, or combinations thereof.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a chromatographic column having a having a mixed-modesize-exclusion chromatography resin with an additional functionality,wherein the mixed-mode size-exclusion chromatography resin can be washedusing a mobile phase containing up to 600 mM total salt concentration.

In one aspect of this embodiment, the mixed mode chromatographic systemcan comprise a chromatographic column having a having a mixed-modesize-exclusion chromatography resin with an additional functionality,wherein the chromatographic column can be washed with a mobile phasewith a flow rate of 0.2 ml/min to 0.4 ml/min.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows represents an example of a system used for quantifyingand/or identifying protein variants using size exclusion chromatographyor ion exchange chromatography.

FIG. 2 shows the Hofmeister series showing the effect of anions andcations on protein precipitation (or promoting hydrophobic interaction).

FIG. 3 shows a mixed-mode size exclusion chromatography massspectrometry system according to an exemplary embodiment.

FIG. 4 shows the extracted ion chromatograms (XIC) obtained onperforming MM-SEC-MS analysis of a digested mixture of bispecificantibody, homodimer 1, and homodimer 2 using mobile phase with differentsalt concentration with a flow rate of 0.3 mL/min according to anexemplary embodiment.

FIG. 5 shows the chart of retention time (minutes) of a protein vs.total salt concentration of the mobile phase for a digested mixture ofbispecific antibody, homodimer 1, and homodimer 2 on performingMM-SEC-MS analysis with a flow rate of 0.3 mL/min according to anexemplary embodiment.

FIG. 6 shows the extracted ion chromatograms (XIC) obtained onperforming MM-SEC-MS analysis of digested mixture of bispecificantibody, homodimer 1, and homodimer 2 using mobile phase with differentsalt concentration with a flow rate of 0.2 mL/min according to anexemplary embodiment.

FIG. 7 shows the chart of retention time (minutes) of a protein vs.total salt concentration of the mobile phase for a digested mixture ofbispecific antibody, homodimer 1, and homodimer 2 on performingMM-SEC-MS analysis with a flow rate of 0.2 mL/min according to anexemplary embodiment.

FIG. 8 shows the effect of concentration of mobile phase on theseparation of digested mixture of F(ab)2 fragments of bispecificantibody, homodimer 1, and homodimer 2 on performing MM-SEC-MS analysisaccording to an exemplary embodiment.

FIG. 9 shows the extracted ion chromatograms (XIC) obtained onperforming MM-SEC-MS analysis of digested and deglycosylated mixture ofan antibody using mobile phase with different salt concentration with aflow rate of 0.2 mL/min according to an exemplary embodiment.

FIG. 10 shows the chart of retention time (minutes) of a protein vs.total salt concentration of the mobile phase for a digested anddeglycosylated mixture of an antibody on performing MM-SEC-MS analysiswith a flow rate of 0.2 mL/min according to an exemplary embodiment.

FIG. 11 shows the effect of concentration of mobile phase on theseparation of digested and deglycosylated mixture of an antibody onperforming MM-SEC-MS analysis with a flow rate of 0.2 mL/min accordingto an exemplary embodiment.

FIG. 12 represents a chart showing a trend in retention time on changingtotal salt concentration when performing MM-SEC-MS analysis according toan exemplary embodiment.

FIG. 13 represents a chart showing a trend in difference in retentiontime on changing total salt concentration when performing MM-SEC-MSanalysis according to an exemplary embodiment.

FIG. 14 shows the extracted ion chromatograms (XIC) obtained onconducting MM-SEC-MS analysis of digested mixture of bispecificantibody, homodimer 1, and homodimer 2 when performing MM-SEC-MS onWaters BEH SEC Column according to an exemplary embodiment.

FIG. 15 shows the chart of retention time (minutes) of a protein vs.total salt concentration of the mobile phase for a digested mixture ofbispecific antibody, homodimer 1, and homodimer 2 when performingMM-SEC-MS analysis on Waters BEH SEC Column according to an exemplaryembodiment.

FIG. 16 shows the relative abundance of a protein vs. retention time(minutes) for a digested and deglycosylated mixture of an antibody onperforming MM-SEC-MS analysis using 40 mM SEC buffer according to anexemplary embodiment.

FIG. 17 shows the chart of retention abundance of a protein vs. mass tocharge ratio of the protein for a digested and deglycosylated mixture ofan antibody on performing MM-SEC-MS analysis according to an exemplaryembodiment.

FIG. 18 shows the relative abundance of a protein vs. retention time(minutes) for a digested and deglycosylated mixture of an antibody onperforming MM-SEC-MS analysis using a native strong-cation-exchangechromatography-mass spectrometry.

FIG. 19 shows the chart of retention abundance of a protein vs. mass tocharge ratio of the protein for a digested and deglycosylated mixture ofan antibody analysis using a native strong-cation-exchangechromatography-mass spectrometry.

FIG. 20 shows quantification of fragments obtained from Asp-N proteasedigestion of an antibody according to an exemplary embodiment.

FIG. 21 shows the susceptibility of an antibody to fragment in vivo byusing identification and quantification of fragments obtained fromtrypsin protease digestion of the antibody according to an exemplaryembodiment.

DETAILED DESCRIPTION

Antibody fragmentation, either enzymatically or non-enzymatically, canform antibody fragments as impurities during the processing of theantibody products. The enormous dynamic proteinaceous species present inprotein-based therapeutics pose a challenge for current massspectrometry-based methods to detect antibody fragments and the site offragmentation since the amount of the antibody fragments may be in lowabundance.

Alternatively, a wide variety of antibody fragments have been designedas therapeutics. The most significant advantages to antibody fragmentsinclude size, manufacturing, tissue penetration, and ability toconcatenate to generate multi-specificity. Sometimes it is useful tostudy or make use of the activity of one portion of an immunoglobulinwithout interference from other portions of the molecule. It is possibleto selectively cleave the immunoglobulin molecule into fragments thathave discrete characteristics. Antibody fragmentation can beaccomplished using reducing agents and proteases that digest or cleavecertain portions of the immunoglobulin protein structure (Nelson (2010)mAbs 2:77-83; 12—Antibody fragments as therapeutics, Editor(s): WilliamR. Strohl, Lila M. Strohl, In Woodhead Publishing Series in Biomedicine,“Therapeutic Antibody Engineering, 2012, 265-595). In order to designand evaluate such antibody fragments, it can be important to identifythe antibody fragments and the site of fragmentation on the antibody bya particular digestive method.

To identify the antibody fragments and the site of fragmentation,traditional separation-based antibody purity assays such aselectrophoresis- and high-performance liquid chromatography (HPLC)-basedmethods lack the needed resolution. Peptide mapping via reverse phaseliquid chromatography (RPLC) coupled with mass spectrometry also hassome limitations as the sample preparation process for RP-LC-MS islengthy, and in some cases the chromatographic conditions such as hightemperature, organic solvents, and acidic pH could induce oxidationartifacts. Hydrophobic interaction chromatography (HIC) and Protein Achromatography also has been used for analysis of antibody oxidation,but can require longer chromatographic run times, and can have a limitedpower for different fragments (Haverick et al. mAbs, (2014) 6:852-858;Boyd et al. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2011)879: 955-960; and Loew et al. J. Pharm. Sci. (2012) 101: 4248-4257).

Additionally, some size exclusion chromatography or ion exchangechromatography methods can be used for separating antibody fragmentsformed on digestion of an antibody. The antibody fragments can furtherbe analyzed using a mass spectrometer or ultraviolet absorbance system.However, the mobile phase from the size exclusion chromatography or ionexchange chromatography column cannot be directly injected into the massspectrometer and requires additional steps including a change in themobile phase (See FIG. 1).

Considering the limitations of existing methods, an effective andefficient method for identification and quantification of antibodyfragments and site of antibody fragmentation using a novel mixedmode—size exclusion chromatography—mass spectrometry system wasdeveloped.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing, particular methods and materials are nowdescribed. All publications mentioned are hereby incorporated byreference.

The term “a” should be understood to mean “at least one”; and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the art;and where ranges are provided, endpoints are included.

Biopharmaceutical products are required to show high levels of potency,purity, and low level of structural heterogeneity. Structuralheterogeneity often affects the bioactivity and efficacy of a drug.Therefore, characterizing and quantifying the therapeutic protein and/orthe impurities is important in pharmaceutical drug development.Structural heterogeneity in a protein can arise from post-translationalmodifications as well as inherent chemical modifications duringmanufacturing and storage conditions. For proteins produced in thebiotechnology industry, complementary separation techniques arenecessary both to purify the target protein and to give an accuratepicture of the quality of the final product. The complexity of theproduct eliminates the use of simple one-dimensional separationstrategies.

As used herein, the term “protein” includes any amino acid polymerhaving covalently linked amide bonds. Proteins comprise one or moreamino acid polymer chains, generally known in the art as “polypeptides”.“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides’ refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known to those of skill in the art. Aprotein may contain one or multiple polypeptides to form a singlefunctioning biomolecule. A protein can include any of bio-therapeuticproteins, recombinant proteins used in research or therapy, trapproteins and other chimeric receptor Fc-fusion proteins, chimericproteins, antibodies, monoclonal antibodies, polyclonal antibodies,human antibodies, and bispecific antibodies. In another exemplaryaspect, a protein can include antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, peptide hormones,and the like. Proteins may be produced using recombinant cell-basedproduction systems, such as the insect bacculovirus system, yeastsystems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHOderivatives like CHO-K1 cells). For a review discussing biotherapeuticproteins and their production, see Ghaderi et al., “Production platformsfor biotherapeutic glycoproteins. Occurrence, impact, and challenges ofnon-human sialylation,” (Biotechnol. Genet. Eng. Rev. (2012) 147-75). Insome exemplary embodiments, proteins comprise modifications, adducts,and other covalently linked moieties. Those modifications, adducts andmoieties include for example avidin, streptavidin, biotin, glycans(e.g., N-acetylgalactosamine, galactose, neuraminic acid,N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG,polyhistidine, FLAGtag, maltose binding protein (MBP), chitin bindingprotein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescentlabels and other dyes, and the like. Proteins can be classified on thebasis of compositions and solubility and can thus include simpleproteins, such as, globular proteins and fibrous proteins; conjugatedproteins, such as, nucleoproteins, glycoproteins, mucoproteins,chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; andderived proteins, such as, primary derived proteins and secondaryderived proteins.

In some exemplary embodiments, the protein can be an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or combinations thereof.

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (C.sub.L1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different exemplaryembodiments, the FRs of the anti-big-ET-1 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs. The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fcfragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAbfragment, a Fd′ fragment, a Fd fragment, and an isolated complementaritydetermining region (CDR) region, as well as triabodies, tetrabodies,linear antibodies, single-chain antibody molecules, and multi specificantibodies formed from antibody fragments. Fv fragments are thecombination of the variable regions of the immunoglobulin heavy andlight chains, and ScFv proteins are recombinant single chain polypeptidemolecules in which immunoglobulin light and heavy chain variable regionsare connected by a peptide linker. An antibody fragment may be producedby various means. For example, an antibody fragment may be enzymaticallyor chemically produced by fragmentation of an intact antibody and/or itmay be recombinantly produced from a gene encoding the partial antibodysequence. Alternatively or additionally, an antibody fragment may bewholly or partially synthetically produced. An antibody fragment mayoptionally comprise a single chain antibody fragment. Alternatively oradditionally, an antibody fragment may comprise multiple chains that arelinked together, for example, by disulfide linkages. An antibodyfragment may optionally comprise a multi-molecular complex.

As used herein, the term “digestion” refers to hydrolysis of the peptidebonds of the proteins. There are several approaches to carrying outdigestion of a protein in a sample using an appropriate hydrolyzingagent, for example, enzymatic digestion or non-enzymatic digestion.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include trypsin, endoproteinase Arg-C,endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), chymotrypsin, pepsin, thermolysin, papain, pronase, and proteasefrom Aspergillus Saitoi. Non-limiting examples of hydrolyzing agentsthat can carry out non-enzymatic digestion include the use of hightemperature, microwave, ultrasound, high pressure, infrared, solvents(non-limiting examples are ethanol and acetonitrile), immobilized enzymedigestion (IMER), magnetic particle immobilized enzymes, and on-chipimmobilized enzymes. For a recent review discussing the availabletechniques for protein digestion see Switazar et al., “ProteinDigestion: An Overview of the Available Techniques and RecentDevelopments” (J. Proteome Research 2013, 12, 1067-1077). One or acombination of hydrolyzing agents can cleave peptide bonds in a proteinor polypeptide, in a sequence-specific manner, generating a predictablecollection of shorter peptides.

One of the widely accepted methods for digestion of proteins in a sampleinvolved the use of proteases. Many proteases are available and each ofthem has their own characteristics in terms of specificity, efficiency,and optimum digestion conditions. Proteases refer to both endopeptidasesand exopeptidases, as classified on the basis of the ability of theprotease to cleave at non-terminal or terminal amino acids within thepeptide. Alternatively, proteases also refer to the six distinctclasses, aspartic, glutamic, and metalloproteases, cysteine, serine, andthreonine proteases, as classified on the mechanism of catalysis. Theterms “protease” and “peptidase” are used interchangeably to refer toenzymes which hydrolyze peptide bonds.

Proteases can also be classified into specific and non-specificproteases. As used herein, the term “specific protease” refers to aprotease with an ability to cleave the peptide substrate at a specificamino acid side chain of a peptide.

As used herein, the term “non-specific protease” refers to a proteasewith a reduced ability to cleave the peptide substrate at a specificamino acid side chain of a peptide. A cleavage preference may be basedon the ratio of the number of a particular amino acid as the site ofcleavage to the total number of cleaved amino acids in the proteinsequences.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.

As used herein, the term “Mixed Mode Chromatography (MMC)” or“multimodal chromatography” includes a chromatographic method in whichsolutes interact with stationary phase through more than one interactionmode or mechanism. MMC can be used as an alternative or complementarytool to traditional reversed-phased (RP), ion exchange (IEX) and normalphase chromatography (NP). Unlike RP, NP and IEX chromatography, inwhich hydrophobic interaction, hydrophilic interaction and ionicinteraction respectively are the dominant interaction modes, mixed-modechromatography can employ a combination of two or more of theseinteraction modes. Mixed mode chromatography media can provide uniqueselectivity that cannot be reproduced by single mode chromatography.Mixed mode chromatography can provide potential cost savings, longercolumn lifetimes and operation flexibility compared to affinity basedmethods.

The phrase “size-exclusion chromatography” or “SEC” or “gel filtration”includes a liquid column chromatographic technique that can sortmolecules according to their size in solution.

As used herein, the terms “SEC chromatography resin” or “SECchromatography media” are used interchangeably herein and can includeany kind of solid phase used in SEC which separates the impurity fromthe desired product (e.g., a homodimer contaminant for a bispecificantibody product). The volume of the resin, the length and diameter ofthe column to be used, as well as the dynamic capacity and flow-rate candepend on several parameters such as the volume of fluid to be treated,concentration of protein in the fluid to be subjected to the process.

As used herein, the term “mixed mode-size exclusion chromatography” or“MM-SEC” can include any chromatographic method which separates proteinsthrough an additional interaction other than the separation based ontheir size. The additional or secondary interaction can exploit one ormore of the following mechanisms: anion exchange, cation exchange,hydrophobic interaction, hydrophilic interaction, charge-chargeinteraction, hydrogen bonding, pi-pi bonding, and metal affinity. Themixed mode-size exclusion chromatography resin can refer to any kind ofsolid phase used for MM-SEC separation. Non-limiting examples are SepaxZenix SEC-300, Waters BEH 300, or Agilent Bio SEC-3.

As used herein, the term “hydrophobic functionality” refers to thehydrophobic interaction of the protein with the SEC chromatographicresin as a secondary interaction. They also significantly impact peakshape, which will have a pronounced effect on the resolving ability ofthe process. Hydrophobic interactions are strongest at high ionicstrength of the mobile phase. For selecting a mobile phase to includehydrophobic functionality in a resin, various ions can be arranged in aso-called soluphobic series depending on whether they promotehydrophobic interactions (salting-out effects) or disrupt the structureof water (chaotropic effect) and lead to the weakening of thehydrophobic interaction (See FIG. 2). Cations can be ranked in terms ofincreasing salting out effect as Ba⁺⁺; Ca⁺⁺; Mg⁺⁺; Li⁺; Cs⁺; Na⁺; K⁺;Rb⁺; NH₄ ⁺, while anions may be ranked in terms of increasing chaotropiceffect as PO⁻; SO₄ ⁻; CH₃CO₂ ⁻; Cl⁻; Br⁻; NO₃ ⁻; ClO₄ ⁻; P; SCN⁻. Ingeneral, Na, K or NH₄ sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr>NaSCN.

As used herein, the term “mass spectrometer” includes a device capableof identifying specific molecular species and measuring their accuratemasses. The term is meant to include any molecular detector into which apolypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer can include three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends heavily on the application.

As used herein, the term “mass analyzer” includes a device that canseparate species, that is, atoms, molecules, or clusters, according totheir mass. Non-liming examples of mass analyzers that could be employedfor fast protein sequencing are time-of-flight (TOF), magnetic/electricsector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap,Fourier transform ion cyclotron resonance (FTICR), and also thetechnique of accelerator mass spectrometry (AMS).

As used herein, the term “tandem mass spectrometry” includes a techniquewhere structural information on sample molecules is obtained by usingmultiple stages of mass selection and mass separation. A prerequisite isthat the sample molecules can be transferred into gas phase and ionizedintact and that they can be induced to fall apart in some predictableand controllable fashion after the first mass selection step. MultistageMS/MS, or MSn, can be performed by first selecting and isolating aprecursor ion (MS²), fragmenting it, isolating a primary fragment ion(MS³), fragmenting it, isolating a secondary fragment (MS⁴), and so onas long as one can obtain meaningful information or the fragment ionsignal is detectable. Tandem MS have been successfully performed with awide variety of analyzer combinations. What analyzers to combine for acertain application is determined by many different factors, such assensitivity, selectivity, and speed, but also size, cost, andavailability. The two major categories of tandem MS methods aretandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers.

Embodiments disclosed herein provide compositions, methods, and systemsfor the rapid characterization of proteins in a sample.

As used herein, the terms “include,” “includes,” and “including,” aremeant to be non-limiting and are understood to mean “comprise,”“comprises,” and “comprising,” respectively.

This disclosure provides methods for quantifying a fragment of anantibody in a sample comprising contacting the sample to achromatographic system having a mixed-mode chromatography resin, washingthe mixed-mode size-exclusion chromatography resin using a mobile phaseto provide an eluent including the fragment, and quantifying thefragment in the eluent using a mass spectrometer.

The disclosure provides methods for identifying a fragment of anantibody in a sample comprising contacting the sample to achromatographic system having a mixed-mode chromatography resin; washingthe mixed-mode chromatography resin using a mobile phase to provide aneluent including the fragment, determining molecular weight of thefragment in the eluent using a mass spectrometer, and correlating themolecular weight data of the fragment to data obtained from at least oneknown protein standard to identify the fragment.

The disclosure also provides methods for identification of a site offragmentation of an antibody comprising contacting a sample includingfragments of an antibody to a chromatographic system having a mixed-modesize-exclusion chromatography resin with an additional functionality,washing the mixed-mode size-exclusion chromatography resin using amobile phase to provide an eluent, determining molecular weight data ofthe fragments of the antibody in said eluent using a mass spectrometer,and correlating the molecular weight data of the fragments to dataobtained from at least one known protein standard.

In some specific exemplary embodiments, the chromatographic system cancomprise a size-exclusion chromatography resin with an additionalinteraction.

In some specific exemplary embodiments, the chromatographic system cancomprise a size-exclusion chromatography resin with hydrophobicinteraction functionality.

In some specific exemplary embodiments, the chromatographic system cancomprise a size-exclusion chromatography resin with charge-chargeinteraction functionality.

In some exemplary embodiments, the fragment can be a digestion productof the antibody. The digestion product can be formed by a hydrolyzingagent. The hydrolyzing agent can include agents carrying out digestionusing enzymatic or non-enzymatic digestion. The hydrolyzing agent can bean agent that can carry out digestion using enzymatic digestion and caninclude trypsin, endoproteinase Arg-C, endoproteinase Asp-N,endoproteinase Glu-C, outer membrane protease T (OmpT),immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS),chymotrypsin, pepsin, thermolysin, papain, pronase, and protease fromAspergillus Saitoi. The hydrolyzing agent can also be an agent that cancarry out digestion using non-enzymatic digestion and can include theuse of high temperature, microwave, ultrasound, high pressure, infrared,solvents. The digestion product can be a product-related impurity.

In some exemplary embodiments, the fragment can include Fab fragment, aFab′ fragment, a F(ab′)2 fragment, a scFv fragment, a Fv fragment, adsFv diabody, a dAb fragment, a Fd′ fragment, a Fd fragment, and anisolated complementarity determining region (CDR) region, triabodies,tetrabodies, linear antibodies, single-chain antibody molecules, andmulti specific antibodies formed from antibody fragments.

In some exemplary embodiments, the antibody can be a protein with a pIin the range of about 4.5 to about 9.0. In one aspect, the antibody canbe a protein with a pI of about 4.5, about 5.0, about 5.5, about 5.6,about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0.

In some exemplary embodiments, the fragment can be a protein with a pIin the range of about 4.5 to about 9.0. in one aspect, the fragment canbe a protein with a pI of about 4.5, about 5.0, about 5.5, about 5.6,about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0.

In one exemplary embodiment, the number of fragments in the sample canbe at least two.

In some exemplary embodiments, amount of total protein in the sampleloaded on the chromatographic system can range from about 10 μg to about100 μg. In one exemplary embodiment, the amount of the sample loaded onthe chromatographic system can be about 10 μg, about 12.5 μg, about 15μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg,about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg,or about 100 μg.

In some exemplary embodiments, the mobile phase used to elute thefragment can be a mobile phase that can be compatible with a massspectrometer. In one aspect, the mobile phase can be ammonium acetate,ammonium bicarbonate, or ammonium formate, or combinations thereof.

In one exemplary embodiment, the total concentration of the mobile phasecan range up to about 600 mM. In one aspect, the total concentration ofthe mobile phase can be about 5 mM, about 6 mM, 7 mM, about 8 mM, 9 mM,about 10 mM, 12.5 mM, about 15 mM, 17.5 mM, about 20 mM, 25 mM, about 30mM, 35 mM, about 40 mM, 45 mM, about 50 mM, 55 mM, about 60 mM, 65 mM,about 70 mM, 75 mM, about 80 mM, 75 mM, about 95 mM, 100 mM, about 110mM, 120 mM, about 130 mM, 140 mM, about 150 mM, 160 mM, about 170 mM,180 mM, about 190 mM, 200 mM, about 225 mM, 250 mM, about 275 mM, 300mM, about 325 mM, 350 mM, about 375 mM, 400 mM, about 4205 mM, 450 mM,about 475 mM, 500 mM, about 525 mM, 550 mM, about 575 mM, or about 600mM.

In some exemplary embodiments, the mobile phase can have a flow rate ofabout 0.1 ml/min to about 0.4 ml/min. In one aspect, the flow rate ofthe mobile phase can be about 0.1 ml/min, about 0.15 ml/min, about 0.20ml/min, about 0.25 ml/min, about 0.30 ml/min, about 0.35 ml/min, orabout 0.4 ml/min.

In one exemplary embodiment, the mass spectrometer can be a tandem massspectrometer.

In another exemplary embodiment, the mass spectrometer can comprise ananospray.

In some exemplary embodiments, the antibody can be a monoclonalantibody.

In some exemplary embodiments, the antibody can be a therapeuticantibody.

In some exemplary embodiments, the antibody can be an immunoglobulinprotein.

In some exemplary embodiments, the antibody can be a bispecificantibody.

In one exemplary embodiment, the bispecific antibody can beAnti-CD20/CD3 monoclonal antibody.

In one exemplary embodiment, the antibody generated using mousefibroblast cell line MG87.

In some exemplary embodiments, the fragment can be an antibody fragmentformed on digestion of the antibody.

In one exemplary embodiment, the fragment can be a post-translationallymodified protein.

In yet another exemplary embodiment, the fragment can be an impurityfound in a biopharmaceutical product.

In another exemplary embodiment, the fragment can be an impurity foundduring the manufacture of the biopharmaceutical product.

In some exemplary embodiments, washing the mixed-mode chromatographyresin using a mobile phase requires less than about 30 minutes. In oneaspect, the time required for washing the mixed-mode chromatographyresin using a mobile phase can be about 10 minutes, about 11 minutes,about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes,about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes,about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes,about 24 minutes, about 25 minutes, about 26 minutes, about 26 minutes,about 27 minutes, about 28 minutes, about 29 minutes, or about 30minutes.

In some exemplary embodiments, the chromatographic system can be usedfor at least about 3 sample runs without cleaning. In one aspect, thechromatographic system can be used for at least about 3 sample runs, atleast about 4 sample runs, at least about 5 sample runs, at least about6 sample runs, at least about 7 sample runs, or at least about 8 sampleruns, without cleaning.

It is understood that the methods are not limited to any of theaforesaid protein, fragment, impurity, and column and that the methodsfor identifying or quantifying may be conducted by any suitable means.

The disclosure also provides a mixed mode chromatographic systemcomprising a chromatographic column 110 capable of being washed using amobile phase to provide an eluent and a mass spectrometer 120 coupled tothe chromatographic column 110 (See FIG. 3).

In one exemplary embodiment, the chromatographic column 110 can becapable of being contacted with a sample including a fragment of anantibody using a sample loading device 100.

In some exemplary embodiments, the amount of the sample that can beloaded on the chromatographic column 110 can range from about 10 μg toabout 100 μg. In one aspect, the amount of the sample that can be loadedon the chromatographic column 110 can be about 10 μg, about 12.5 μg,about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg,about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about95 μg, or about 100 μg.

In some exemplary embodiments, the chromatographic column 110 can becapable of being washed with a mobile phase. In one aspect, the mobilephase can be ammonium acetate, ammonium bicarbonate, or ammoniumformate, or combinations thereof.

In one exemplary embodiment, the total concentration of the mobile phasethat can be used with the chromatographic column 110 can range up toabout 600 mM. In one aspect, the total concentration of the mobile phasethat can be used with the chromatographic column 110 can be about 5 mM,about 6 mM, 7 mM, about 8 mM, 9 mM, about 10 mM, 12.5 mM, about 15 mM,17.5 mM, about 20 mM, 25 mM, about 30 mM, 35 mM, about 40 mM, 45 mM,about 50 mM, 55 mM, about 60 mM, 65 mM, about 70 mM, 75 mM, about 80 mM,75 mM, about 95 mM, 100 mM, about 100 mM, 120 mM, about 130 mM, 140 mM,about 150 mM, 160 mM, about 170 mM, 180 mM, about 190 mM, 200 mM, about225 mM, 250 mM, about 275 mM, 300 mM, about 325 mM, 350 mM, about 375mM, 400 mM, about 425 mM, 450 mM, about 475 mM, 500 mM, about 525 mM,550 mM, about 575 mM, or about 600 mM.

In another exemplary embodiment, the mobile phase that can be used withthe chromatographic column 110 can have a flow rate of 0.1 ml/min to 0.4ml/min. In one aspect, the flow rate of the mobile phase that can beused with the chromatographic column 110 can be about 0.1 ml/min, about0.15 ml/min, about 0.20 ml/min, about 0.25 ml/min, about 0.30 ml/min,about 0.35 ml/min, or about 0.4 ml/min.

In one exemplary embodiment, the mobile phase used with thechromatographic column 110 capable of being contacted with a sampleincluding a fragment of an antibody, can be used to elute the fragment.

In some exemplary embodiments, the chromatographic column 110 can becapable of being coupled with a mass spectrometer 120.

In one exemplary embodiment, the mass spectrometer 120 can comprise ananospray.

In some exemplary embodiments, the mass spectrometer 120 can be a tandemmass spectrometer.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment (asillustrated in FIG. 3). The fragment can include Fab fragment, a Fab′fragment, a F(ab′)2 fragment, a scFv fragment, a Fv fragment, a dsFvdiabody, a dAb fragment, a Fd′ fragment, a Fd fragment, and an isolatedcomplementarity determining region (CDR) region, triabodies,tetrabodies, linear antibodies, single-chain antibody molecules, andmulti specific antibodies formed from antibody fragments.

In one exemplary embodiment, the mixed mode chromatographic system canbe used to identify 140 and/or quantify 130 more than one fragments.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of amonoclonal antibody.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of atherapeutic antibody.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of animmunoglobulin protein.

In one exemplary embodiment, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of anIgG1 protein.

In one exemplary embodiment, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of anIgG4 protein.

In one exemplary embodiment, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of abispecific antibody.

In one exemplary embodiment, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of anAnti-CD20/CD3 monoclonal antibody.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment of anantibody fragment formed on digestion of the antibody.

In yet another exemplary embodiment, the mixed mode chromatographicsystem can be capable of identifying 140 and/or quantifying 130 afragment which can be an impurity found in a biopharmaceutical product.

In another exemplary embodiment, the mixed mode chromatographic systemcan be capable of identifying 140 and/or quantifying 130 a fragment,wherein the fragment can be an impurity found during the manufacture ofthe biopharmaceutical product.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment, whereinthe fragment can be a protein with a pI in the range of about 4.5 toabout 9.0.

In some exemplary embodiments, the mixed mode chromatographic system canbe capable of identifying 140 and/or quantifying 130 a fragment, whereinthe fragment can be a product-related impurity.

In one exemplary embodiment, the number of fragments in the sample canbe at least two.

In some exemplary embodiments, the chromatographic column 110 capable ofbeing used for at least about 3 sample runs without cleaning.

In one exemplary embodiment, the chromatographic column 110 can be usedfor at least about 3 sample runs, at least about 4 sample runs, at leastabout 5 sample runs, at least about 6 sample runs, at least about 7sample runs, or at least about 8 sample runs, without cleaning.

It is understood that the system is not limited to any of the aforesaidprotein, impurity, mobile phase, or chromatographic column.

The consecutive labeling of method steps as provided herein with numbersand/or letters is not meant to limit the method or any embodimentsthereof to the particular indicated order.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is incorporated by reference, in its entirety and for allpurposes, herein.

The disclosure will be more fully understood by reference to thefollowing Examples, which are provided to describe the disclosure ingreater detail. They are intended to illustrate and should not beconstrued as limiting the scope of the disclosure.

EXAMPLES Example 1. Mixed Mode Size Exclusion Chromatography Coupled toMass Spectrometry (MM-SEC-MS)

Separation was performed by an Acquity system (Waters, Milford, Mass.,USA) coupled to a UV detector and an electrospray mass spectrometer(Thermo Exactive EMR, USA).]. The mass spectrometer was operated in thepositive resolution mode and data were recorded from m/z [2000-15,000].Calibration was achieved on the acquisition range according tomanufacturer's procedure.

Example 2. Detection of Fragments in a Digested Mixture of a BispecificAntibody, Homodimer 1 and Homodimer 2 Using MM-SEC-MS on Zenix-SECColumn

2.1 Sample Preparation of Bispecific Antibody

The anti-CD20×anti-CD3 Bispecific Antibody is a hinge-stabilizedCD20×CD3 bispecific full-length antibody (Ab) based on an IgG4 isotypemodified to reduce Fc binding. It was designed to bind T cells (via CD3)and CD20-expressing cells. The Bispecific Antibody was produced byfollowing the methodology as described by Smith et al. (Sci. Rep. (2015)5:17943).

2.2 Generation of the Fragments of Bispecific Antibody, Homodimer 1 andHomodimer 2

1 mg of Bispecific antibody was digested with 100 units FabRICATOR® for60 minutes in 0.1 M Tris-HCl buffer pH 7.5 at 37° C.

2.3 MM-SEC-MS

The analysis using MM-SEC-MS was performed isocratically using a ZenixSEC-300 MK column (4.6×300 nm, 3 μm) on the system as described inExample 1. Elution was monitored by UV at 280 nm.

Eight sets of experiments were carried out wherein the totalconcentration of the mobile phase was varied: 10 mM buffer, 20 mMbuffer, 30 mM buffer, 40 mM buffer, 50 mM buffer, 60 mM buffer, 70 mMbuffer, and 75 mM buffer. The elution was carried out at a flow rate of0.3 mL/min. The chromatography was run on Waters Acquity I-class UPLCsystem with the column temperature of room temperature. Theequilibration was performed using the mobile phase composed of ammoniumacetate (buffer A) and ammonium bicarbonate (buffer B) at 14:1 molarratio.

For analytical runs, the injection loads consisted of 10 μg of the totalprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The mass spectrometry data was analyzed by using Intact Masssoftware.

An increased separation between the fragments was observed when lowerconcentration of mobile phase was used (e.g., for 10 mM bufferconcentration, a significant separation between the F(ab)2 fragment ofBispecific Ab, F(ab)2 fragment of Homodimer 1, and Fc fragment wasobserved (See FIG. 4). Lower salt concentrations enhance thecharge-charge interaction in the MM-SEC column, which provided a betterseparation between the fragments for the Bispecific Ab (See FIGS. 4 and5).

Example 3. Detection of Fragments in a Digested Mixture of a BispecificAntibody, Homodimer 1 and Homodimer 2 Using MM-SEC-MS on Zenix-SECColumn

3.1 Sample preparation of bispecific antibody and generation of thefragments of Bispecific antibody, homodimer 1 and homodimer was carriedout as illustrated in 2.1 and 2.2.

3.2 MM-SEC-MS

The analysis using MM-SEC-MS was performed isocratically using a ZenixSEC-300 MK column (4.6×300 nm, 3 μm) on the system as described inExample 1. Elution was monitored by UV at 280 nm.

Six sets of experiments were carried out wherein the total concentrationof the mobile phase was varied: 50 mM buffer, 60 mM buffer, 70 mMbuffer, 75 mM buffer, 100 mM buffer, and 300 mM buffer. The elution wascarried out at a flow rate of 0.2 mL/min. The chromatography was run onWaters Acquity I-class UPLC system with the column temperature of roomtemperature. The equilibration was performed using the mobile phase.

For analytical runs, the injection loads consisted of 10 μg of the totalprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The mass spectrometry data was analyzed by using Intact Masssoftware from Protein Metrics.

Lower salt concentrations enhance the charge-charge interaction in theMM-SEC column and higher salt concentrations enhance the hydrophobicinteraction in the MM-SEC column. At 50 mM buffer concentration, thesystem showed a better separation between the F(ab)2 fragment of thebispecific antibody and the Fc fragment, whereas at 300 mM, the systemshowed a better separation between the F(ab)2 fragment of the bispecificantibody and the F(ab)2 fragment of the homodimer 1 (See FIGS. 6 and 7).The difference in retention times of the F(ab)2 fragments for bispecificantibody and homodimer 1 and of the F(ab)2 fragments for bispecificantibody and homodimer 2 further shows that a better separation wasattained at high buffer concentration of 300 mM (FIG. 8).

Example 4. Detection of Fragments of an Antibody Molecule (Ab1) UsingZenix SEC-300, 3 μm, 300 Å, 7.8×300 mm

4.1 Generation of the Fragments of Ab1

0.5 mg of Ab1 was digested with [50 unit] FabRICATOR® for 60 minutes in0.1 M Tris-HCl buffer pH 7.5 at 37° C. to form fragments.

4.2 MM-SEC-MS

The analysis using MM-SEC-MS was performed isocratically using a ZenixSEC-300 MK column (4.6×300 nm, 3 μm) on the system as described inExample 1. Elution was monitored by UV at 280 nm.

Five sets of experiments were carried out wherein the totalconcentration of the mobile phase was varied: 30 mM buffer, 40 mMbuffer, 66 mM buffer, 100 mM buffer, and 200 mM buffer. The elution wascarried out at a flow rate of 0.2 mL/min. The chromatography was run onWaters Acquity I-Class UPLC system with the column temperature of roomtemperature. The equilibration was performed using the mobile phase.

For analytical runs, the injection loads consisted of 10 μg of the totalprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The mass spectrometry data was analyzed by using Intact Masssoftware from Protein Metrics.

The runs with mobile phases of differing concentration revealed thatlower salt concentration enhancing the charge-charge interaction in theMM-SEC column providing better separation of the fragments (See FIGS. 9and 10). Comparing the retention times of Fc fragment of Ab1 and F(ab)2fragment of Ab1, the mobile phase concentration of 30 mM provides thebest separation. Further, the difference in retention time of the HChomodimer-F(ab)2 and HC*CDR3 clipping product further shows that abetter separation was attained at the low buffer concentration of 30 mM(FIG. 11).

The fragments as illustrated in examples 1-3 show better separation atdifferent buffer concentrations. This effect could be due to differenttypes of interaction: charge, shape, or hydrophobicity of the proteinswith the size exclusion chromatography resin used. The charge on theprotein at a given salt concentration depends on pI values (Tables 1 and2). Significant differences in charge interactions with the MM-SEC mediaare obtained with larger differences in pI values. For the same class ofIgG molecules, differences in hydrophobicity originate from the Fabregion. At lower salt concentrations, retention time can be driven bycharge-charge interaction and at higher salt concentrations, retentionis driven by hydrophobic interaction. Thus, acidic or hydrophobicmolecules can be separated by using mobile phase with higher saltconcentration in the MM-SEC-MS system and basic molecules can beseparated by using mobile phase with lower salt concentration in theMM-SEC-MS system (See FIG. 12 and FIG. 13).

TABLE 1 Intact F(ab)2 Fc isotype mAb molecule pI MW pI MW pI IgG4Bispecific Ab 7.66 145.337 8.32 97,827 5.81 homodimer 1 7.28 144,6778.13 98,491 5.77 homodimer 2 8.03 145,998 8.48 97,164 5.86

TABLE 2 Intact F(ab)2 Fc isotype mAb molecule pI MW pI MW pI IgG4 Ab17.59 145,544 8.29 98,052 5.81 Ab1 HC/HC homodimer 6.57 145,948 6.9898,445 5.77

Example 5. Detection of Fragments in a Digested Mixture of a BispecificAntibody, Homodimer 1 and Homodimer 2 Using MM-SEC-MS on Waters BEH SECColumn

5.1 Sample Preparation of Bispecific Antibody and Generation of theFragments of Bispecific Antibody, Homodimer 1 and Homodimer was CarriedOut as Illustrated in 2.1 and 2.2.

5.2 MM-SEC-MS

The analysis using MM-SEC-MS was performed isocratically using a WatersBEH SEC column (4.6×300 nm, 3 μm) on the system as described inExample 1. Elution was monitored by UV at 280 nm.

Eight sets of experiments were carried out wherein the totalconcentration of the mobile phase was varied: 20 mM buffer, 27.6 mMbuffer, 30 mM buffer, 40 mM buffer, 50 mM buffer, 100 mM buffer, 150 mMbuffer, and 300 mM buffer. The elution was carried out at a flow rate of0.2 mL/min. The chromatography was run on Waters Acquity I-class UPLCsystem with the column temperature of room temperature. Theequilibration was performed using the mobile phase.

For analytical runs, the injection loads consisted of 10 μg of the totalprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The mass spectrometry data was analyzed by using Intact Masssoftware.

Similar to the process carried on a Zenix SEC column, MM-SEC-MS analysisof digested mixture of bispecific Ab, homodimer 1, and homodimer 2exhibited larger separations at lower concentration of buffer, i.e., 20mM buffer (See FIGS. 14 and 15).

Example 6. Identification of a New HC*CDR3 Clipping Site in Ab1 UsingMM-SEC-MS

6.1 Generation of the Fragments of Ab1

The fragments of Ab1 were generated using the methodology as describe in4.1

6.2 MM-SEC-MS

The analysis using MM-SEC-MS was performed isocratically using a ZenixSEC column (4.6×300 nm, 3 μm) on the system as described in Example 1.Elution was monitored by UV at 280 nm.

The analysis was carried out using a mobile phase with totalconcentration of 40 mM buffer and the elution was carried out at a flowrate of 0.2 mL/min. The chromatography was run on Waters Acquity I-classUPLC system with the column temperature of room temperature. Theequilibration was performed using the mobile phase.

For analytical runs, the injection loads consisted of 10 μg of the totalprotein. The elution was carried out using an isocratic gradientconsisting of ammonium acetate (buffer A) and ammonium bicarbonate(buffer B). The mass spectrometry data was analyzed by using Intact Masssoftware from Protein Metrics. The total ion chromatogram for the sampleshowed two peaks with retention times of 16 minutes and 17 minutes. Themass analysis of one of the peaks had fragments with mass to chargeratio of 5162.62 and 6663.59 (See FIG. 16). This peak at 16 min showedtwo m/z peak distributions, one with one of the charge state being5162.62 corresponds to the amide bond hydrolysis between K105 and F106but the fragments are still held together by non-covalent interactions,the other m/z peak distributions (one of the charge state being m/z6663.59) correspond to the dissociated fragment from the hydrolysisproduct. The other peak showed a fragment with mass to charge ratio of5161.43 (FIGS. 16 and 17). The masses were compared to the calculationsbased on the Ab1 sequence and identified to be the Bispecific Ab-F(ab)2with one clipping site (amide bond hydrolysis) (MW=98,069.8), BispecificAb-F(ab)2 missing HC*(E1-K105) (MW=86,612.7), and intact Bispecific AbF(ab)2 (MW=98,048.1). These fragments led to identification the site iffragmentation in the HC of the Bispecific Ab: between Lysine105 andPhenylalanine106.

6.3 Confirmation of the HC*CDR3 Clipping Site by Native SCX-MS

The analysis using strong cation chromatography was performed using aYMC BioPro SP-F column (4.6×100 nm). Elution was monitored by UV at 280nm. Separation was performed by an Acquity system (Waters, Milford,Mass., USA) coupled to a UV detector and an electrospray massspectrometer (Thermo Exactive EMR, USA). The mass spectrometer wasoperated in the positive resolution mode and data were recorded from m/z2000-15,000. Calibration was achieved on the acquisition range accordingto manufacturer's procedure.

The analysis was carried out using gradient elution at a flow rate of 4mL/min: 100% A to 100% B in 18 min; wherein solvent A was 20 mM ammoniumacetate, pH 5.6 and solvent B was 140 mM ammonium acetate+10 mM ammoniumbicarbonate. The equilibration was performed using the mobile phase.

For analytical runs, the injection loads consisted of 50 μg of the totalprotein. The elution was carried as describe above. The massspectrometry data was analyzed by using Intact Mass software fromProtein Metrics. The chromatogram for the sample showed two peaks withretention times of 12 minutes and 13 minutes. The mass analysis of oneof the peaks had fragments with mass to charge ratio of 5162.66 and6663.51. The other peak showed a fragment with mass to charge ratio of5161.51 (FIGS. 18 and 19), which confirmed the fragments obtained byusing the MM-SEC-MS system: Bispecific Ab-F(ab)2 with one clipping site(amide bond hydrolysis) (MW=98,069.8), Bispecific Ab-F(ab)2 missingHC*(E1-K105) (MW=86,612.7), and intact Bispecific Ab F(ab)2(MW=98,048.1).

6.4 Quantitation of the HC*CDR3 Clipping Fragment

100 ug of HC*CDR3 clipping fragment was digested with 2 ug AspN enzymefor 18 hours in 0.1 M Tris-HCl buffer pH 7.5 at 37° C. to form itsfragments. The chromatogram of the fragment using PepMap column provideda quantification of the HC*CDR3 clipping fragment of Ab1 (FIG. 20).

6.5 Susceptibility of Ab1 to Plasma Proteases to Form HC*CDR3 ClippingFragment In Vivo

MM-SEC-MS analysis of digested fragments formed by was performedisocratically using a Zenix SEC-300 MK column (4.6×300 nm, 3 μm) on thesystem as described in Example 1. Elution was monitored by UV at 280 nm.Trypsin enzyme was used to predict the susceptibility of Ab1 in vivo toplasma proteases to form the HC*CDR3 Clipping fragment (identified inexample 6.2). To the digested fragments of Ab1 antibody as described in4.1, trypsin was added in the ratio of 200:1 at 37° C.

An injection load consisting of 10 μg of the total protein was loaded onthe column. The analysis was performed using a mobile phase with totalconcentration of 30 mM (ammonium acetate (buffer A) and ammoniumbicarbonate) and the elution was carried out at a flow rate of 0.2mL/min. The chromatography was run on Waters Acquity I-Class UPLC systemwith the column temperature of room temperature.

At time zero of addition of the trypsin enzyme, the fragmentation showeda presence of HC*CDR3 Clipping fragment (FIG. 21, upper panel). Onsimilar analysis at time equal to 35 minutes after addition of thetrypsin enzyme, the fragmentation showed a significant increase inpresence of HC*CDR3 clipping fragment (FIG. 21, lower panel). Thisindicated that the antibody Ab1 is cleaved at the site (K105-F106) bytrypsin, suggesting that the antibody Ab1 in vivo is susceptible to sucha cleavage in vivo.

What is claimed is:
 1. A method for identification of a site offragmentation of an antibody, said method comprising: contacting asample including at least one fragment of the antibody to achromatographic system having a mixed-mode size-exclusion chromatographyresin with an additional functionality; washing said mixed-modesize-exclusion chromatography resin using a mobile phase to provide aneluent with the at least one fragment of the antibody; determiningmolecular weight data of the at least one fragment of the antibody insaid eluent using a mass spectrometer; and correlating the molecularweight data of the at least one fragment of the antibody to dataobtained from at least one known protein standard.
 2. The method ofclaim 1, wherein the mobile phase has ammonium acetate, ammoniumbicarbonate, or ammonium formate, or combinations thereof.
 3. The methodof claim 1, wherein the mobile phase has a total concentration of lessthan about 600 mM of ammonium acetate and ammonium bicarbonate.
 4. Themethod of claim 1, wherein the mobile phase has a flow rate of about 0.2ml/min-about 0.4 ml/min.
 5. The method of claim 1, wherein the amount ofthe sample loaded onto the mixed-mode size-exclusion chromatographyresin is about 10 μg to about 100 μg.
 6. The method of claim 1, whereinthe antibody is a monoclonal antibody.
 7. The method of claim 1, whereinthe antibody is a bispecific antibody.
 8. The method of claim 1, whereinthe antibody is a therapeutic antibody.
 9. The method of claim 1,wherein the mass spectrometer is coupled to the chromatographic system.10. The method of claim 1, wherein the site of fragmentation of theantibody is in hinge region of the antibody.
 11. The method of claim 1,wherein the site of fragmentation of the antibody is in a constantimmunoglobulin domain.
 12. The method of claim 1, wherein the site offragmentation of the antibody is in a variable domain of the antibody.13. The method of claim 1, wherein the additional functionality is acharge-charge interaction functionality.
 14. The method of claim 1,wherein the additional functionality is a hydrophobic interactionfunctionality.
 15. The method of claim 1, wherein the at least onefragment is a F(ab)2 fragment.
 16. The method of claim 1, wherein the atleast one fragment is be a Fc fragment.
 17. A method for identificationof a fragment of an antibody, said method comprising: contacting asample including the fragment of the antibody to a chromatographicsystem having a mixed-mode size-exclusion chromatography resin withhydrophobic functionality; washing said mixed-mode size-exclusionchromatography resin using a mobile phase to provide an eluent with thefragment of the antibody; determining molecular weight data of thefragment of the antibody in said eluent using a mass spectrometer; andcorrelating the molecular weight data of the fragment of the antibody todata obtained from at least one known protein standard to identify thefragment.
 18. A method for quantification of a fragment of an antibody,said method comprising: contacting a sample including the fragment ofthe antibody to a chromatographic system having a mixed-modesize-exclusion chromatography resin with hydrophobic functionality;washing said mixed-mode size-exclusion chromatography resin using amobile phase to provide an eluent with the fragment of the antibody; andquantifying the amount of the fragment of the antibody.